It is well known that racemic mixtures of N-acyl D,L-amino acids can be resolved by the use of mold acylase enzyme (U.S. Pat. No. 3,907,638). In this process, water, N-acyl D,L-amino acid and acylase are stirred in organic solvent at a pH and temperature suitable for acylase activity. After reaction, the acylase is in the aqueous phase, the optically active N-acyl amino acid and the acid formed by deacylation are in the organic phase and the desired deacylated amino acid is in the aqueous phase as solute or precipitated as a solid. These procedures are effective for N-acyl amino acids using the N-acyl group as the means for resolution. This patent does not teach resolution of racemic mixtures of carbonyl substituted amino acids which are not N-acyl substituted.
It is also known that esters of amino acids with large hydrophobic side chains attached to the alpha carbon atom such as L-phenylalanine can be hydrolyzed by various enzymes, such as chymotrypsin. The specificity of the enzyme for hydrolyzing the ester of the L-isomer forms the basis of a process for the enzymatic resolution of racemic mixtures of phenylalanine (U.S. Pat. No. 3,813,317 and the references cited therein as well as U.S. Pat. No. 3,878,043). In U.S. Pat. No. 3,813,317, an enzyme such as chymotrypsin is used to resolve an aqueous racemic mixture of ring substituted phenylalanines. The reaction is carried out in aqueous solution. A balance had to be struck between a low concentration of ester needed for good enzyme activity and a high concentration needed for easier product recovery.
In U.S. Pat. No. 3,878,043, an intermediate of L-DOPA is resolved into the L-isomer by hydrolysis of the ester group. The hydrolysis is conducted in an aqueous medium preferably including a water soluble organic solvent such as an alkanol. The D-isomer product is separated by contacting the aqueous solution with an organic solvent which takes up the D-isomer. The aqueous solution is then concentrated and the L-isomer separated.
This method of resolving amino acid esters suffers from several inherent problems. During the course of the hydrolysis, acid is liberated from the newly generated carboxyl group. As the enzyme activity is pH sensitive, the acid must be continuously neutralized to prevent the pH from dropping sufficiently low to seriously effect enzyme activity. The D-amino acid ester acts as an inhibitor to the enzyme used in the hydrolysis reaction. As the resolution proceeds, the concentration of the D-amino acid ester increases relative to the L-ester, further decreasing enzyme activity.
For an industrial process, it is desirable to process high concentrations of racemic mixtures of amino acid esters. However, it is known that high concentrations of such amino acid esters i.e. phenylalanine, can be catalyzed by the enzyme to form insoluble peptides. These peptides lower product purity and yields.